Biological Sciences Division Research Highlights

Chemical Probe Finds Fungal Organism Function

Aspergillus fumigatus is a common fungus. Most people breathe in its spores every day without being affected; however, in people with lung diseases or weakened immune systems, it causes the disease Aspergillosis. Photo courtesy of the Centers for Disease Control

Results: Two unique chemical probes
designed at Pacific Northwest National Laboratory are helping scientists find
how a pathogenic organism responsible for a severe lung infection thrives in
human serum. These probes use multiplexed activity-based protein profiling (ABPP), which revealed
significant changes in Aspergillus fumigatus metabolism and stress
response when placed in culture with human serum over time.

Why It Matters. A. fumigatus spores are ubiquitous in the atmosphere, and everybody inhales an
estimated several hundred spores each day. But for people whose immune response
is compromised from taking immunosuppressive drugs, by irradiation or malnutrition,
or by diseases such as cancer or AIDS, this opportunistic pathogen is responsible
for pulmonary invasive aspergillosis (IA). Patients with IA are usually
critically ill, and the disease is difficult to cure.

The
PNNL team hypothesized that A. fumigatus
uses human serum, the clear fluid that remains
after blood is allowed to clot, as a nutrient and that enzyme activity within the serum impacts
the organism's metabolism, nutrient sensing, and scavenging response within an
immunocompromised host environment. They simultaneously used two novel activity-based chemical probes they designed and constructed to target the reactivity of A. fumigatus during growth. The team found
that probe-protein reactivity changes in the presence or absence of human
serum.

The
information provides valuable insight into how A. fumigatus survives in a host environment on a fundamental level.
During the course of IA, the fungus' filamentous
structure breaches host tissue and interacts with serum, where it readily grows
because of its unique ability to extract iron from human transferrin in an
iron-limited environment. Furthermore,
A. fumigatus can use serum proteins as
building blocks for growth, but the full effect of serum on its cellular
processes and its relevance to disease are not fully understood.

"We are demonstrating that we can compare two
systems or one organism under multiple conditions to tease out more information
about protein regulation," said Dr. Susan Wiedner, a
PNNL Linus Pauling Distinguished Postdoctoral Fellow, and lead author of the
paper published in The Journal of
Biological Chemistry.

The scientists wanted to determine which
proteins interact with a small-molecule activity-based probe. "We found that
under two different growth conditions, the number and identity of proteins that
interact with the probes change drastically," said Wiedner. "We could measure the
abundance of probe-labeled proteins by liquid chromatography-mass spectrometry
(LC-MS) -based proteomics and see profiles of labeled proteins based on growth
condition."

Methods: Various approaches to LC-MS-based
proteomics have emerged.In a
typical global analysis, thousands of proteins are measured from a complex
proteome. However, by using ABPP, scientists can target a subset of a few hundred
proteins from the complex proteome.

"ABPP is a more directed approach than using
global proteomics," said Wiedner. "We can look at the difference of probe-labeled
protein abundances among various systems and conditions. This then tells us
more about the system's biology, such as the differences between probe-reactivity
of metabolic proteins. Some fungal proteins interact more with the probe in the
presence of human serum, which tells us something about what metabolism might be
doing under those conditions." A direct comparison of a global analysis and an
ABPP analysis showed differences in measured proteins detected by ABPP that were
not detected by global analysis.

In turn, this can lead to more in-depth
studies such as generating gene knock-out mutants and performing enzymatic
assays, all of which could be used to develop effective treatments and
detection of IA.

Over the last two decades, ABPP development
has been a growing but still-small field. PNNL has one of the groups working on
this, led by PNNL chemist Dr. Aaron Wright, senior author on this paper.

What's Next: Currently, PNNL scientists are
developing ABPP probes and supporting bioinformatics capabilities to measure
cellulose degradation in microbial communities. ABPP
can be useful for a variety of things including target validation of drug
candidates and protein inhibitor discovery.

Said Wright, "Probes can compete with known
drugs for protein-binding sites, which results in drug target and drug
off-target validation. Some probes are broad, like those used in this study.
But some can be very selective for an enzyme class. We can design the probe
based on the enzyme class being targeted. We used a multiplexed approach here, where
two probes were used simultaneously to target more than one type of protein.
Previous studies only use one ABP at a time for proteome analysis."

Acknowledgments

Sponsors: The work was funded by the National Institutes of Health's National Center for Research
Resources and National Institute of General Medical Sciences, and PNNL's Laboratory
Directed Research and Development Program. The work used instrumentation and
capabilities developed under support from NIH and the U.S. Department of Energy
Office of Biological and Environmental Research (DOE-BER). Work was performed
in the Environmental Molecular Sciences Laboratory, a DOE-BER national
scientific user facility at PNNL. Widener was supported by PNNL's Linus
Pauling Distinguished Postdoctoral Fellowship.

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More about activity-based probes

A key development
in identifying active proteins is activity-based protein profiling (ABPP), an
emergent technology in the chemical proteomics field, which studies how
molecules bind to proteins. In the graphic shown in the text, probes consisting of an
electrophilic reactive group, a targeting group, and a reporter tag are made
via organic chemistry, shown (A) as a line joining a triangle, oval, and
rectangle. These Activity-Based Probes (ABPs) are then added to proteome samples or live cells, shown here as crescent-shaped
proteins, where the probe then covalently binds to specific regions
of a protein. These regions could be the active site of an enzyme or a
ligand-binding site.

(B) A reporter
group (green circle) is added to the protein via click chemistry-followed by
enrichment. Then the probe and protein are pulled out of the sample using
affinity purification (strong interaction), and all the grey proteins are
washed away.

(C) The remaining
proteins and probe are then prepped for mass spectrometry analysis. Ideally,
orange proteins will only interact with the probe if they are active. Proteins
that are inhibitor-bound or do not contain a nucleophilic* amino acid residue
won't interact with the ABP.

This methodology
fills critical knowledge gaps that can't be determined from genome technologies
that only detect and quantify macromolecules (RNA, protein, metabolites);
namely, the status and identity of active enzymes or of probe-reactive
proteins. Instead, ABPP relies on chemical probes that directly target a subset,
usually via enzyme mechanism-based inhibition, of reactive proteins.

*Of a chemical species that donates an
electron pair to an electrophile to form a chemical bond in a reaction.